Microparticles comprising a sulphur-containing compound

ABSTRACT

The present invention provides microparticles comprising a sulphur-containing compound, such as cysteamine, or a pharmaceutically acceptable salt, hydrate or ester thereof. Also provided is a composition comprising the microparticles and a stabilizing agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage filing under 35 U.S.C. § 371of International Application No. PCT/GB2017/051637 filed Jun. 6, 2017entitled “MICROPARTICLES COMPRISING A SULPHUR-CONTAINING COMPOUND,”which claims priority of U.S. Provisional Application No. 62/346,969filed Jun. 7, 2016 and G.B. Application No. 1609940.0 filed Jun. 7,2016, the disclosures of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to microparticles comprising asulphur-containing compound, such as cysteamine or cystamine, or apharmaceutically acceptable salt, hydrate or ester thereof.

BACKGROUND TO THE INVENTION

Cystic fibrosis (CF) is a multisystem disorder caused by mutations inthe cystic fibrosis transmembrane conductance regulator (CFTR) gene,located on chromosome.

Lung disease remains the leading cause of morbidity and mortality inpatients with CF [Davis P B, Drumm M, Konstan M W. Cystic fibrosis. Am JRespir Crit Care Med 1996; 154:1229; Goss C H, Rosenfeld M. Update oncystic fibrosis epidemiology; Curr Opin Pulm Med 2004; 10:510; Brennan AL, Geddes D M. Cystic fibrosis. Curr Opin Infect Dis 2002; 15:175;Gibson R L, Burns J L, Ramsey B W. Pathophysiology and management ofpulmonary infections in cystic fibrosis. Am J Respir Crit Care Med 2003;168:918.

One of the major drivers of CF lung disease is infection (Sagel S D,Gibson R L, Emerson J, et al. Impact of Pseudomonas and Staphylococcusinfection on inflammation and clinical status in young children withcystic fibrosis. J Pediatr 2009; 154:183; Cystic Fibrosis FoundationAnnual Patient Registry 2013. Available at:http://www.cff.org/research/ClinicalResearch/PatientRegistryReport/(Accessedon Aug. 7, 2015).

The approach to treating infection in CF is multifaceted, involvingantibiotics, chest physiotherapy, inhaled medications to promotesecretion clearance, and anti-inflammatory agents. Undoubtedly, improveduse of antibiotics is responsible for a substantial portion of theincreased survival that has occurred in patients with CF (Brennan A L,Geddes D M. Cystic fibrosis. Curr Opin Infect Dis 2002; 15:175; Sagel SD, Gibson R L, Emerson J, et al. Impact of Pseudomonas andStaphylococcus infection on inflammation and clinical status in youngchildren with cystic fibrosis. J Pediatr 2009; 154:183).

There remains a need for better therapies for treating and preventinglung diseases/conditions in particular those associated with mucous-richenvironments such as the CF lung. In addition there remains a need tolimit the amount or doses of antibiotics used with the introduction ofnovel, replacement therapies or adjunct treatments that can improve theeffectiveness of currently available treatments in the treatment orprevention of bacterial infections, in particular in the CF lung.

Surprisingly, we have shown that microparticles provide a useful mode ofdelivery for cysteamine to patients with lung disease.

STATEMENTS OF THE INVENTION

According to a first aspect of the present invention there is provided amicroparticle or microparticles comprising a sulphur-containingcompound, or a pharmaceutically acceptable salt, hydrate or esterthereof.

As used herein “sulphur-containing compound” is intended to covercysteamine, cystamine or a derivative thereof. The sulphur-containingcompound may be an aminothiol. Examples of aminothiols includecysteamine and derivatives thereof. The term “derivative thereof” mayencompass 2-methylthio ethylamine (cinnamate), 2-methyl thio ethylurea,N-(2-methylthio ethyl) p-acetamido benzamide, 2-aminoethanethiol,N-(2-methylthio ethyl)p-acetamido benzenesulfonamide,N-(2-propylthioethyl)-p-methoxy benzamide, N-(butylthio ethyl)nicotinamide, N-(2-dodecylthio ethyl) p-butoxybenzamide, N-(2-methylthioethyl) p-toluenesulfonamide, N-(2-isopropylthio ethyl) propionamide.N-(2-octylthio ethyl) acetamide, N-(2-butylthio ethyl)methanesulfonamide, N-(2-isopentylthioethyl)butane, bis 1,4-(2-acetamidoethylthio), 2,3-butanediol, 2-hexadecylthio ethylamine hydrochloride,2-allylthio ethylamine malate, 9-octadecene 2-ylthio ethylaminehydrochloride, 2-dodecylthio ethylamine hydrochloride, 2-isopentylthioethylamine mandelate, 2-octadecylthio ethylamine salicylate,2-.beta.-hydroxyethyl thio ethylurea, 2-.beta.-hydroxyethylthioethylamine hydrochloride, 2-(2,3-dihydroxy propylthio)ethylaminep-toluenesulfonate, 2-(2-hydroxypropylthio)elhylamineoxalate,N-(2-methylthio ethyl (phenylacetamide, 2-(2,2-dimethoxy ethylthio)ethylamine hydrochloride, 2-(2,2-dimethoxy ethylthio)ethylamineundecylenate, 2-(2,2-diethoxy ethylthio) ethylamineundecylenate, 2-(2,2-diethoxy ethylthio)ethylamine acetate,2-undecenylthio ethylamine, 2-.beta.-ureidoethylthio ethylaminehydrochloride, 2-.beta.-acetamidoethylthio ethylamine tropate, 2,2′-thiodiethylamine fumarate, 2,2′-thio diethylurea, 3-.beta.-aminoethylthiopropylamine hydrochloride, S-.beta.-ureidoethyl thiocarbamate,2-ethoxycarbonylthio ethylamine hydrochloride, 2-dimethylaminocarbonylthio ethylamine sulfate, 2-butoxycarbonyl methylthio ethylurea,2-ethyloxycarbonylmethylthio ethylamine hydrochloride,6-.beta.-aminoethylthio hexanoate of methyl hydrochloride,5-.beta.-aminoethylthio pentanoic acid, 2-phenylthio ethylaminedihydrogen phosphate, 2-p-t-butylphenylthio ethylamine trichloracetate,2-p-methoxyphenylthio ethylamine ditartrate, 2-tolylthio ethylaminehydrobromide, 2-(1-biphenyl thio) ethylamine hydrochloride,2-N-pentachlorophenylthio ethyl acetamide, 2-benzylthio ethylaminemalate, 2-benzylthio ethylamine nicotinate, 2-benzylthio 2-methylpropylamine hydrochloride, 2-benzylthio propylamine lactate,N-(2-benzylthio ethyl)nicotinamide hydrochloride, N-(2-benzylthio ethyl)10-undecene amide, N-(2-benzylthio ethyl) hexadecanamide,S-.beta.-aminoethyl mercaptobutyric acid, N-(2-benzylthioethyl)formamide, N-(2-benzylthio ethyl)phenyl acetamide,N-[2-(2,6-dimethyl phenyl)ethyl] hexanamide, 2-o-aminophenylthioethylamine succinate, N-(2-benzylthio ethyl) glutamine,S-.beta.-aminoethyl mercapto acetic acid (3-S-.beta.-aminoethyl)mercapto propionic acid, (3-S-.gamma.-amino propyl) mercapto aceticacid, S(2-p-methoxybenzamido ethyl) mercapto 2-(2-naphtyl methylthio)ethylamine hydrochloride, 2-(2-naphtyl methylthio) ethylaminedisuccinate, (2-thenyl) 2-thio ethylamine hydrobromide, 2-N-acetyl(2-thenylthio-ethylamine, 2-o-chlorobenzylthio ethylamine hydrochloride,2-p-chlorobenzylthio ethylamine glycolate, 2-o-fluorobenzylthioethylamine hydrochloride, 2-furfurylthio ethylamine hydrochloride,2-tetrahydrofurfurylthio ethylamine p-amino-benzoate, 2-.beta.-phenylethylthio ethylamine glutamate, 2-diphenyl methylthio ethylaminehydrochloride, 2-triphenyl methylthio ethylamine hydrochloridehemihydrate, 2-(2-pyridylethylthio)ethylamine hydrochloride,2-(2-p-toluene sulfonamido ethylthio) pyridine N-oxide,2-.beta.-aminoethylthiomethyl pyridine N-oxide dihydrochloride,2-.beta.-aminoethylthio pyridine N-oxide hydrochloride, 2,4-dichloro2-benzylthio ethylamine aspartate, N-[2-(3,4-dichloro benzylthio)ethyl]butyramide, N-[2-(2,6-dichloro benzylthio)ethyl] dodecanamide,N-[2-(3,5-dichloro benzylthio)ethyl] trifluoroacetamide hydrochloride,2-p-ethoxybenzylthio ethylamine hydrochloride, N-[2-m-fluorobenzylthioethyl] chloroacetamide, 2-p-bromobenzylthio ethylamine succinate,2-(3,4-dimethoxy benzylthio)ethylamine malate, 2-(3,4-methylenedioxybenzylthio)ethylamine hydrochloride, 2-(2,4-dichlorocetylthio)ethylamine, 2(3,4,5-trimethoxy benzylthio)ethylaminehydrocinnamate, 2-p-methoxy benzylthio ethylamine salicylate,2-o-methylbenzylthio ethylamine phenyl-acetate,N-[2-p-dimethylaminobenzylthio ethyl] methane-sulfonamide,2-p-phenoxybenzylthio ethyl amine hydrochloride, 2-.beta.-aminoethylthiopyridine hydrochloride, 2-benzylthio ethylamine citrate, N-[2-benzylthioethyl] 2,4-dihydroxy 3,3-dimethyl butyramide, N-(2-benzylthio ethyl)6,8-dihydroxy 7,7-dimethyl 5-oxo 4-aza octanamide, N-[2-(2-pyridylthio)ethyl] propionamide, 2-(2-pyridyl methylthio)ethylaminedihydrochloride, 2-benzylthio ethylamine pantothenate,S-(.beta.-acetamidoethyl)mercapto acetate of beta.-morpholinoethyl,S-(.beta.-phenyl acetamidoethyl)mercapto acetate N′-methyl 2-piperazinoethyl, S-(.beta.-ureidoethyl)mercaptoacetate of beta.-pyrrolidino-ethy,S-(.beta.-trifluoroacetamidoethyl)-.beta.mercapto-propionate of.beta.-dimethyl aminoethyl, 2-p-nitrobenzylthio ethylamine crotonate,2-.beta.-morpholinocarbonyl ethylthio ethylamine hydrochloride, N,N-di(hydroxyethyl) S-(.beta.-benzamido-ethyl) mercapto-acetamido, N[2-N′-methyl piperazino carbonylthio ethyl] acetamide, 2-(1-naphthylthio)ethylamine hydrochloride, N-(3-.beta.-ureidoethylthio propyl)succinamic acid, 3-allylthio propylamine, 3-(2,2′-dimethoxyethylthio)propylamine, 3-(2,2′-dimethoxy ethylthio)propyl amine sulfate,S-.beta.-aminoethyl mercapto acetic acid, the hydrochloride ofS-.beta.-aminoethyl mercapto acetic acid,N-(2-benzylthioethyl)acetamide, N-(2-benzylthioethyl)propionamide,N-(2-benzylthioethyl)butyramide, N-(2-benzylthioethyl)methanesulfonamide, N-(2-benzylthioethyl)ethanesulfonamide,N-(2-benzylthioethyl-propane sulfonamide,N-(2-benzylthioethyl)butanesulfonamide,S-(2-p-acetamidobenzenesulfonamido ethyl) mercapto acetic acid,S-(2-p-acetamidobenzamido ethyl) mercapto acetic acid,N-(2-thenylthioethyl)acetamide, 2-b enzylthio propylamine, 2-benzylthio2-methyl propyl amine, 2-(2-p-toluenesulfonamido ethylthio) pyridineN-oxide, S-(2-p-butoxybenzamidoethyl)mercapto acetic acid, 2-t-butylthioethylamine hydrochloride, 2-methoxycarbonylmethylthio ethylaminehydrochloride, 2-ethoxycarbonylmethylthio ethylamine hydrochloride,2-propoxycarbonylmethyl thio ethylamine hydrochloride,2-butoxycarbonylmethylthio ethyl amine hydrochloride, 2,2′-thiodiethylamine dihydrochloride, 3-(2-aminoethylthio)alanine hydrochloride,2-benzylthio ethyl ammonium diacid phosphate, 2-methylthio ethylamine,N-(methylthio ethyl) p-acetamidobenzamide,N-(2-methylthioethyl)nicotinamide, N-(2-methylthioethyl)benzamide,N-(2-methylthioethyl) p-butoxybenzamide, N-(2-methylthioethyl)butyramide, N-(2-methylthioethyl) propionamide, N-(2-methylthioethyl)acetamide, N-(2-methylthioethyl) butane sulfonamide,N-(2-octylthioethyl) methane sulfonamide, 2-cetylthio ethylaminehydrochloride, 2-(2-hydroxyethylthio) ethylamine hydrochloride,2-methylthio ethyl amine phenyl acetate and 2-methylthio ethylamineundecylenate.

Alternatively, the sulphur-containing compound may be an organicdisulphide, such as cystamine.

The sulphur-containing compound of the invention may be administered inthe form of pharmaceutically acceptable salts. The pharmaceuticallyacceptable salts of the present invention can be synthesized from theparent compound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17thed., Mack Publishing Company, Easton, Pa., US, 1985, p. 1418, thedisclosure of which is hereby incorporated by reference; see also Stahlet al, Eds, “Handbook of Pharmaceutical Salts Properties Selection andUse”, Verlag Helvetica Chimica Acta and Wiley-VCH, 2002. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings or, as the case may be, an animalwithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The invention thus includes pharmaceutically-acceptable salts of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof for example the conventional non-toxic saltsor the quaternary ammonium salts which are formed, e.g., from inorganicor organic acids or bases. Examples of such acid addition salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base saltsinclude ammonium salts, alkali metal salts such as sodium and potassiumsalts, alkaline earth metal salts such as calcium and magnesium salts,salts with organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine,lysine, and so forth. Also, the basic nitrogen-containing groups may bequaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethyl bromidesand others.

In a preferred aspect of the invention, the microparticles have particlesize of about 0.5 to 15 microns, for example 1 to 13 microns, including4 to 8 microns. Particle size may be defined as “volume mean diameter”and as such the microparticles may have a volume mean diameter of about0.5 to 15 microns, for example 1 to 13 microns, including 4 to 8microns. The microparticles may have a volume mean diameter of 2 to 4microns/micrometers (2-4 μm).

Mean is a calculated value similar to the concept of average. Thevarious mean calculations are defined in several standard documents (ISO9276-2:2001: Representation of results of particle size analysis—Part 2:Calculation of average particle sizes/diameters and moments fromparticle size distributions; ASTM E 799-03 Standard Practice forDetermining Data Criteria and Processing for Liquid Drop Size Analysis).There are multiple definitions for mean because the mean value isassociated with the basis of the distribution calculation (number,surface, volume), see (TN154, Particle Size Result Interpretation:Number vs. Volume Distributions, available atwww.horiba.com/us/particle) for an explanation of number, surface, andvolume distributions. The equation for defining the volume mean is shownbelow. The best way to think about this calculation is to think of ahistogram table showing the upper and lower limits of n size channelsalong with the percent within this channel. The D_(i) value for eachchannel is the geometric mean, the square root of upper×lower diameters.For the numerator take the geometric D_(i) to the fourth powermultiplied by the percent in that channel, summed over all channels. Forthe denominator take the geometric D_(i) to the third power multipliedby the percent in that channel, summed over all channels.

${D\left\lbrack {4,3} \right\rbrack} = \frac{\sum\limits_{1}^{n}\;{D_{i}^{4}v_{i}}}{\sum\limits_{1}^{n}\;{D_{i}^{3}v_{i}}}$

The volume mean diameter has several names including D4,3 or D50/D90.

As used herein, the terms “diameter” or “d” in reference to particlesrefers to the number average particle size, unless otherwise specified.An example of an equation that can be used to describe the numberaverage particle size is shown below:

$d = \frac{\sum\limits_{i = 1}^{p}\;{n_{i}d_{i}}}{\sum\limits_{i = 1}^{p}\; n_{i}}$where n=number of particles of a given diameter (d).

As used herein, the terms “geometric size”, “geometric diameter, “volumeaverage size”, “volume average diameter” or “dg” refers to the volumeweighted diameter average. An example of equations that can be used todescribe the volume average diameter is shown below:

$d_{g} = \left\lbrack \frac{\sum\limits_{i = 1}^{p}\;{n_{i}d_{i}^{\; 3}}}{\sum\limits_{i = 1}^{p}\; n_{i}} \right\rbrack^{1/3}$where n=number of particles of a given diameter (d).

As used herein, the term “volume median” refers to the median diametervalue of the “volume-weighted” distribution. The median is the diameterfor which 505 of the total are smaller and 50% are larger andcorresponds to a cumulative fraction of 50%.

Geometric particle size analysis can be performed on a Coulter counter,by light scattering, by light microscopy, scanning electron microscopy,or transmittance electron microscopy, as known in the art. It is agenerally held belief that the ideal scenario for delivery to the lungis to have an aerodynamic diameter <5 micrometers. See, e.g., Edwards etal., J Appl. Physiol. 85(2):379-85 (1998); Suarez & Hickey, Respir.Care. 45(6):652-66 (2000).

As used herein, the term “aerodynamic diameter” refers to the equivalentdiameter of a sphere with density of 1 g/mL were it to fall undergravity with the same velocity as the particle analysed. The aerodynamicdiameter (d_(a)) of a microparticle is related to the geometric diameter(dg) and the envelope density (p_(e)) by the following:da=dg√{square root over (ρe)}

Porosity affects envelope density which in turn affects aerodynamicdiameter. Thus porosity can be used to affect both where themicroparticles go in the lung and the rate at which the microparticlesrelease the pharmaceutical agent in the lung. Gravitational settling(sedimentation), inertial impaction, Brownian diffusion, interceptionand electrostatic affect particle deposition in the lungs.

The microparticles may have an aerodynamic diameter of about 0.5 to 15microns, for example 1 to 13 microns, including 4 to 8 microns. Themicroparticles may have an aerodynamic diameter of 2 to 4microns/micrometers (2-4 μm).

In a further aspect, the invention provides a composition comprisingmicroparticles according to the first aspect of the invention and astabilizing agent. In some instances the stabilizing agent is selectedfrom the group consisting of monosaccharides, disaccharides,trisaccharides, oligosaccharides and their corresponding sugar alcohols,polysaccharides and chemically modified carbohydrates.

In a yet further aspect, the invention provides a composition comprisinga sulphur-containing compound, or a pharmaceutically acceptable salt,hydrate or ester thereof, and a stabilizing agent as defined herein.

The stabilizing agent may be a sugar such as trehalose.

The stabilising agent may be a sugar alcohol selected from the groupconsisting of lactose, erythritol, ribitol, xylitol, galactitol,glucitol and mannitol. Preferably the stabilising agent is mannitol.

In a preferred composition of the invention, the composition comprisesup to 20%w/w sulphur-containing compound, for example between 1 and 15%such as between about 5 and 10% w/w Sulphur-containing compound.Typically, the composition comprises about 5 or 10% Sulphur-containingcompound.

As used herein, the term “about” is intended to vary the specifiedamount to allow for minor fluctuations of between + or 10% of thespecified amount.

In a preferred composition of the invention, the composition comprisesup to 85% stabilising agent. The composition may comprise between 80 and95% w/w stabilizing agent, for example between 85 and 90% w/wstabilizing agent. Typically, the composition comprises about 90%stabilizing agent.

It has been shown that in compositions according to the invention whichcomprise trehalose or mannitol, cysteamine has increased formulationstability.

In a preferred embodiment of the invention the sulphur-containingcompound is cysteamine or cystamine, preferably cysteamine. In a furtherembodiment of the invention, the sulphur-containing compound iscysteamine bitartrate.

In a preferred embodiment the composition is provided as an aqueouscomposition.

The composition of the invention may further comprise leucine. Leucinehas surprisingly been shown to improve the stability of the formulation.In one embodiment of the invention, the composition comprises between 1and 10% leucine, preferably about 5% leucine.

The composition may be in a solid dose form selected from the groupconsisting of microparticles, microspheres, and powders. Preferably thecomposition is provided as a dry powder. The powder may containparticles having a geometric diameter of about 3 to 8 microns, including4 to 8 microns, such as 3 to 7 microns. In one embodiment, the powdercontains particles having a geometric diameter of up to about 5 microns,for example 2 to 4 microns.

A further aspect of the invention provides microparticles according tothe first aspect of the invention, or a composition according to theinvention, for use in the treatment or prevention of lung disease.

A yet further aspect of the present invention relates to a method oftreating or preventing lung disease comprising administeringmicroparticles according to the first aspect of the invention, or acomposition according to the invention, to a subject suffering, orhaving previously suffered from, from lung disease.

As used herein the term “lung disease” includes any disease or conditionof the lung including cystic fibrosis, specifically lung infectionsassociated with cystic fibrosis, and chronic obstructive pulmonarydisease (COPD). COPD is the name for a collection of lung diseasesincluding chronic bronchitis, bronchiectasis, emphysema and chronicobstructive airways disease. The term lung disease is also intended toinclude any respiratory disease which has a mucous or infectiouselement, for example a chronic cough, common cold, influenza,hantavirus, pneumonia and pleurisy.

A further aspect of the invention provides a therapeutic composition (orcombination) which may be useful in the treatment or prevention of lungdisease, which comprises microparticles according to the first aspect ofthe invention, or a composition according to the invention, and at leastone additional pharmaceutical agent. The additional pharmaceutical agentmay be selected from the group consisting of antimicrobial agents suchas antiviral, antifungal or antibacterial agents e.g. antibiotics,mucolytic agents, vasodilators such as bronchidilators, antihypertensiveagents, cardiovascular drugs and calcium channel blockers. Preferablythe additional pharmaceutical agent is an antibiotic.

The term “antibiotic” is used to refer to antibacterial agents that maybe derived from bacterial sources. Antibiotic agents may be bactericidaland/or bacteriostatic.

The antibiotic agent may contain a β-lactam ring. The β-lactam ring ispart of the core structure of several antibiotic families, the principalones being the penicillins, cephalosporins, carbapenems, andmonobactams. These antibiotic agent are called β-lactam antibiotics.

Generally the antibiotic agent is of the group consisting ofaminoglycosides, ansamycins, carbacephem, β-lactams carbapenems,cephalosporins, (including first, second, third, fourth and fifthgeneration cephalosporins), penicillin, monobactams), glycylcyclines,lincosamides, lipopeptides, macrolides, nitrofurans, oxazolidinones,quinolones, sulfonamides, polypeptides and tetracyclins.

The antibiotic agent may be of the group consisting of aminoglycosides,ansamycins, carbacephem, carbapenems, cephalosporins (including first,second, third, fourth and fifth generation cephalosporins),lincosamides, macrolides, monobactams, nitrofurans, quinolones,penicillin, sulfonamides, polypeptides and tetracyclins. Alternativelyor additionally the antibiotic agent may be effective againstmycobacteria.

The antibiotic agent may be an aminoglycoside such as Amikacin,Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin or Paromomycin.

The antibiotic agent may be an http://en.wikipedia.org/wiki/Ansamycinsuch as Geldanamycin and Herbimycin

Alternatively the antibiotic agent may be a carbacephem such asLoracarbef.

The antibiotic agent is a carbapenem such as Ertapenem, Doripenem,Imipenem/Cilastatin or Meropenem.

Alternatively the antibiotic agent may be a cephalosporins (firstgeneration) such as Cefadroxil, Cefazolin, Cefalexin, Cefalotin orCefalothin, or alternatively a Cephalosporins (second generation) suchas Cefaclor, Cefamandole, Cefoxitin, Cefprozil or Cefuroxime.Alternatively the antibiotic agent may be a Cephalosporins (thirdgeneration) such as Cefixime, Cefdinir, Cefditoren, Cefoperazone,Cefotaxime, Cefpodoxime, Ceftibuten, Ceftizoxime and Ceftriaxone or aCephalosporins (fourth generation) such as Cefepime and Ceftobiprole.

The antibiotic agent may be a lincosamides such as Clindamycin andAzithromycin, or a macrolide such as Azithromycin, Clarithromycin,Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin,Telithromycin and Spectinomycin.

Alternatively the antibiotic agent may be a monobactams such asAztreonam, or a nitrofuran such as Furazolidone or Nitrofurantoin.

The antibiotic agent may be a penicillin such as Amoxicillin,Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin,Flucloxacillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G or V,Piperacillin, Temocillin and Ticarcillin.

The antibiotic agent may be an oxazolidinone such as linezolid ortedizolid.

The antibiotic agent may be a sulfonamide such as Mafenide,Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silversulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide,Sulfasalazine, Sulfisoxazole, Trimethoprim, andTrimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX).

The antibiotic agent may be a quinolone such as Ciprofloxacin, Enoxacin,Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid,Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin andTemafloxacin.

The antibiotic agent may be a polypeptide. Examples of such polypeptidesinclude Bacitracin, Colistin and Polymyxin B. In one embodiment, theantibiotic agent is not a polypeptide.

The antibiotic agent may be a lipopeptide. Examples of such lipopeptidesinclude Daptomycin and Surfactin.

Alternatively, the antibiotic agent may be a tetracycline such asDemeclocycline, Doxycycline, Minocycline and Oxytetracycline

Alternatively the antibiotic agent may be a glycylcycline. Examples ofsuch glycylcyclines include tigecycline.

Alternatively or additionally the antibiotic agent may be effectiveagainst mycobacteria. In particular the antibiotic agent may beClofazimine, Lamprene, Dapsone, Capreomycin, Cycloserine, Ethambutol,Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentineor Streptomycin.

In one embodiment, the antibiotic agent is a macrolide and/or anaminoglycoside and/or sulphonamides.

In one embodiment, the antibiotic is selected from tobramycin,azithromycin, telithromycin, ciproflaxin, ceftazidime.

In one embodiment, the antibiotic agent is not ciproflaxin. In anotherembodiment the antibiotic is not tobramycin.

The antibiotic agent may be active in the treatment or prophylaxis ofinfections caused by Enterobacteriaceae (e.g. E. coli or Klebsiellaspp., such as K. pneumoniae) or non-Enterobacteriaceae bacteria such asBurkholderia spp.

Generally the antibiotic agent is active in the treatment or prophylaxisof infections caused by gram-negative or gram-positive bacteria, such asPseudomonas spp.

In one embodiment of the invention, the antibiotic is not a β-lactamantibiotic.

The active agents of the invention may be provided as pharmaceuticalcompositions additionally containing one or more pharmaceuticallyacceptable diluents, excipients and/or carriers. For example, theadditional pharmaceutical agent may be provided as a compositioncomprising the agent and a carrier such as lactose or mannitol.

In a preferred aspect of the invention, the microparticles, or thecomposition, according to the invention, and the additionalpharmaceutical agent may be administered simultaneously, sequentially orseparately. The microparticles, or composition, and additionalpharmaceutical agent may be provided as a combination package. Thecombination package may further comprise instructions for simultaneous,separate or sequential administration of each of the microparticles, orcomposition, and additional pharmaceutical agent. For sequentialadministration, the microparticles, or composition, and additionalpharmaceutical agent can be administered in any order.

The at least one additional pharmaceutical agent may be provided inmicroparticles distinct from said microparticles of the first aspect ofthe invention. Alternatively, the at least one additional pharmaceuticalagent may be provided in a form other than microparticles.

In one embodiment of the invention, the microparticles of the firstaspect of the invention, or composition according to the invention,comprise the at least one additional pharmaceutical agent.

In a further embodiment of the invention, the at least one additionalpharmaceutical agent is administered in microparticles distinct from themicroparticles of the first aspect, or composition, of the invention.

In a yet further embodiment of the invention, the at least oneadditional pharmaceutical agent is administered in a form other thanmicroparticles.

In one embodiment, the microparticles or composition of the inventioncomprising a sulphur-containing compound, such as cysteamine, and/or anadditional pharmaceutical agent to be administered in addition to thesulphur-containing compound have a volume average diameter between 0.1and 5 micrometers (e.g., between 1 and 5 micrometers, between 2 and 5micrometers, etc.). In another embodiment, the microparticles orcomposition of the invention, and/or an additional pharmaceutical agent,have a volume average diameter of up to 10 micrometers, for targetingdelivery to the large bronchi. Particle size (geometric diameter andaerodynamic diameter) is selected to provide an easily dispersed powderthat upon aerosolization and inhalation readily deposits at a targetedsite in the respiratory tract (e.g., upper airway, deep lung, etc.),preferably while avoiding or minimizing excessive deposition of theparticles in the oropharyngel or nasal regions. In one preferredembodiment, the porous microparticles have a volume average diameter ofbetween 2 and 5 micrometers, for example between 2 and 4 micrometers.

Considerable attention has been devoted to the design of therapeuticaerosol inhalers to improve the efficiency of inhalation therapies.Timsina et. al., Int. J Pharm., 101: 1-13 (1995); and Tansey, I. P.,Spray Technol. Market, 4: 26-29 (1994). Attention has also been given tothe design of dry powder aerosol surface texture, regarding particularlythe need to avoid particle aggregation, a phenomenon which considerablydiminishes the efficiency of inhalation therapies. French, D. L.,Edwards, D. A. and Niven, R. W., J. Aerosol ScL, 27: 769-783 (1996). Drypowder formulations (“DPFs”) with large particle size have improvedflowability characteristics, such as less aggregation (Visser, J.,Powder Technology 58: 1-10 (1989)), easier aerosolization, andpotentially less phagocytosis. Rudt, S. and R. H. Muller, J. ControlledRelease, 22: 263-272 (1992); Tabata, Y. and Y. Bcada, J. Biomed. Mater.Res., 22: 837-858 (1988). Dry powder aerosols for inhalation therapy aregenerally produced with mean geometric diameters primarily in the rangeof less than 5 micrometers. Ganderton, D., J Biopharmaceutical Sciences,3: 101-105 (1992); and Gonda, I. “Physico-Chemical Principles in AerosolDelivery,” in Topics in Pharmaceutical Sciences 1991, Crommelin, D. J.and K. K. Midha, Eds., Medpharm Scientific Publishers, Stuttgart, pp.95-115, 1992. Large “carrier” particles (containing no drug) have beenco-delivered with therapeutic aerosols to aid in achieving efficientaerosolization among other possible benefits. French, D. L., Edwards, D.A. and Niven, R. W., J. Aerosol ScL, 27: 769-783 (1996).

Drugs currently administered by inhalation come primarily as liquidaerosol formulations. However, many drugs and excipients, especiallyproteins, peptides (Liu, R., et al., Biotechnol. Bioeng., 37: 177-184(1991)), and biodegradable carriers such as poly(lactide-co-glycolides)(PLGA), are unstable in aqueous environments for extended periods oftime. This can make storage as a liquid formulation problematic. Inaddition, protein denaturation can occur during aerosolization withliquid formulations. Considering these and other limitations, dry powderformulations (DPF's) are gaining increased interest as aerosolformulations for pulmonary delivery. Darnms, B. and W. Bains, NatureBiotechnology (1996); Kobayashi, S., et al, Pharm. Res., 13(1): 80-83(1996); and Timsina, M., et al., hit. J. Pharm., 101: 1-13 (1994).However, among the disadvantages of DPF's is that powders of ultrafineparticulates usually have poor flowability and aerosolizationproperties, leading to relatively low respirable fractions of aerosol,which are the fractions of inhaled aerosol that escape deposition in themouth and throat. Gonda, I., in Topics in Pharmaceutical Sciences 1991,D. Crommelin and K. Midha, Editors, Stuttgart: Medpharm ScientificPublishers, 95-117 (1992). A primary concern with many aerosols isparticulate aggregation caused by particle-particle interactions, suchas hydrophobic, electrostatic, and capillary interactions. The presentinvention aims to address these issues.

Thus, in a further aspect, the invention provides an inhalation devicecomprising microparticles of the first aspect, or composition, of theinvention. The device may be selected from a dry powder inhalationdevice and a metered dose inhaler.

In a further aspect of the invention the composition is obtained bypreparing an aqueous solution of microparticles, or sulfhydryl (SH)compound, and stabilising agent and evaporating the water from thesolution. Preferably the evaporating step is by spray drying.

Thus, a further aspect of the invention provides a process for preparinga composition according to the invention comprising preparing an aqueoussolution of microparticles, or sulfhydryl (SH) compound, and stabilisingagent and evaporating water from the aqueous solution. Preferably theevaporating step is by spray drying.

The microparticles according to the invention may be in the form of adry powder. The microparticles may release an effective amount of asulfhydryl (SH) compound, over a duration of at least two hours frominhalation of said microparticles by a human subject. In a preferredembodiment, substantially all of the Sulphur-containing compound isreleased by 24 hours from inhalation of said microparticles by a humansubject.

Microparticles are convenient to administer, thereby enhancing theextent of patient compliance. The microparticles, or composition, of theinvention may be administered in a single puff. Alternatively, themicroparticles are formulated to provide sustained release ofcysteamine. The microparticles may facilitate local delivery ofcysteamine to the lungs or systemic delivery via the lungs.

The microparticles, or compositions, of the invention may also beadministered intranasally or by inhalation and may be delivered in theform of a dry powder inhaler or an aerosol spray presentation from apressurised container, pump, spray, atomiser, nebuliser, with or withoutthe use of a suitable propellant. Preferably the microparticles, orcompositions, of the invention are administered to the respiratorytract.

As used herein, the terms “comprise,” “comprising,” “include,” and“including” are intended to be open, non-limiting terms, unless thecontrary is expressly indicated.

The invention will now be described by way of example only withreference to the following figures:

FIG. 1 is a graph showing Particle size distribution in batch 57#08a;

FIG. 2 is a graph showing Particle size distribution in batch 57#08b;

FIG. 3 is a graph showing Particle size distribution in batch 57#07(placebo);

FIG. 4. Lynovex/lactose study demonstrating reduction in Pseudomonaslung burden;

FIG. 5. Lynovex (cysteamine) and Tobramycin combination resulting inreduced lung burden;

FIG. 6. Mouse weight does not reduce in the presence of a combination ofLynovex and Tobramycin.

EXAMPLES Example 1: Spray Drying as a Potential Formulation Techniquefor the Delivery of Cysteamine Bitartrate by Oral Inhalation

Materials

Cysteamine Bitartrate: Manufactured by Recordati, batch number 140514-1was supplied by Nova Biotics.

Oleic Acid: Fluka, 75096-1L, lot number BCBN9185V.

Water: Deionized, Millipore, RiOs 5 system, serial number F8HN7 8491K.

L-Leucine: Sigma, L-8000, lot number 91k0906.

Trehalose: Sigma, T9449-1006, Lot number 011M7000N.

Methods:

Initial spray drying studies using solutions of cysteamine bitartrateformulated with oleic acid and trehalose

Several batches of cysteamine bitartrate were produced by spray dryingsolutions containing the active ingredient alone and with addedtrehalose and oleic acid (added as a potential taste masking agent).

Cysteaminine bitartrate was allowed to warm to room temperature for 30minutes before opening. For each batch to be spray dried, 100 mgcysteamine bitartrate powder was added to 10 ml deionised water, to givea total solids concentration of 1% w/v. This was stirred until fullydissolved.

Additional excipients (oleic acid and trehalose) were added to thecysteamine bitartrate solution to assess their impact on the powderproperties after spray drying. The solutions were spray dried using aBuchi B290 spray dryer, fitted with a high-efficiency cyclone and aBuchi two-fluid nozzle. Full spray drying conditions are given in Table1 below:

TABLE 1 Spray Drying Conditions Results from these initial studiesconfirmed that the presence of oleic acid in the formulation led to poorpowder properties and low recoveries. Aspirator 100% Liquid Feed Rate 2ml/minute Atomisation Pressure 5.5 bar Inlet temperature See Table 1Outlet temperature See Table 1

A summary of the batches spray dried is described below in Table 2.

TABLE 2 Production of Initial Feasibility Batches Containing Oleic AcidInitial spray drying studies using solutions of cysteamine bitartrateformulated with trehalose (no oleic acid) Spray Dryer Batch Component AComponent B Component C Solvent Result Temp 052#053 Cysteamine Oleicacid N/A EtOH:Water, Waxy, Inlet: 155° C. Bitartrate 5% 2:1 glassy solidOutlet: 83° C. 95% deposited on the walls of the cyclone 052#055Cysteamine Oleic acid N/A EtOH:Water, Waxy, Inlet: 78° C. Bitartrate 5%2:1 glassy solid Outlet: 48° C. 95% deposited on the walls of thecyclone 052#056 Cysteamine Oleic acid Trehalose EtOH:Water, Waxy, Inlet:75° C. Bitartrate 5% 25% 2:1 glassy solid Outlet: 46° C. 70% depositedon the walls of the cyclone 052#057 Cysteamine Oleic acid TrehaloseEtOH:Water, Waxy, Inlet: 63° C. Bitartrate 1.7% 65.7% 2:1 glassy solidOutlet: 40° C. 32.6% deposited on the walls of the cyclone 052#058Cysteamine Oleic acid N/A Ethyl Acetate:Water, Waxy, Inlet: 50° C.Bitartrate 5% 5:1 glassy solid Outlet: 36° C. 95% deposited on the wallsof the cyclone 052#059 Cysteamine Oleic acid N/A Water:Ethyl Waxy,Inlet: 50° C. Bitartrate 5% acetate glassy solid Outlet: 38° C. 95%(added to deposited on crystals of the walls of API are the cycloneformed)

Based on the spray drying results obtained in 3.1 (below) it was decidedto remove oleic acid from the formulation.

Cysteaminine bitartrate was allowed to warm to room temperature for 30minutes before opening. For each batch to be spray dried, 100 mgcysteamine bitartrate powder was added to 10 ml deionised water, to givea total solids concentration of 1% w/v. This was stirred until fullydissolved.

Trehalose was added to the cysteamine bitartrate solution to assess itsimpact on the properties of the spray dried powder. The solutions werespray dried using a Buchi B290 spray dryer, fitted with ahigh-efficiency cyclone and a Buchi two-fluid nozzle. Full spray dryingconditions are given in Table 3 below.

TABLE 3 Spray Drying Conditions Aspirator 100% Liquid Feed Rate 2ml/minute Atomisation Pressure 5.5 bar Inlet temperature See Table 4Outlet temperature See Table 4

A summary of the batches spray dried is described below in Table 4below.

TABLE 4 Spray Drying Conditions for Trehalose Formulations Spray dryingcysteamine bitartrate formulated with Trehalose and L-Leucine (spraydried batch number 052#155, 052#140, 052#121 with Leucine 052#122without Leucine) Spray Dryer Batch Component A Component B Component CSolvent Result Temp 052#060* Cysteamine Trehalose N/A Water Whitepowder. Inlet: 81° C. Bitartrate 90% Outlet: 42° C. 10% 052#062Cysteamine Trehalose N/A Water Waxy, glassy Inlet: 82° C. Bitartrate 50%solid Outlet: 44° C. 50% deposited on the walls of the cyclone 052#063Cysteamine Trehalose N/A Water Dry white Inlet: 114° C. Bitartrate 75%powder. Outlet: 61° C. 25% 052#064 Cysteamine Trehalose N/A Water Drywhite Inlet: 136° C. Bitartrate 75% powder. Outlet: 71° C. 25% 052#65Cysteamine Trehalose N/A Water Dry white Inlet: 162° C. Bitartrate 75%powder. Outlet: 79° C. 25% 052#66 Cysteamine Trehalose N/A Water Wetlooking Inlet: 148° C. Bitartrate 65% powder. Not Outlet: 70° C. 35%free-flowing. 52#67 Cysteamine Trehalose N/A Water Damp looking Inlet:147° C. Bitartrate 70% powder. Outlet: 72° C. 30% Forms aggregates.052#097* Cysteamine Trehalose N/A Water Dry white Inlet: 121° C.Bitartrate 75% powder. Outlet: 71° C. 25% Spray pressure 5.5 Bar *Usedto generate additional data

In order to further improve the properties of the spray dried powder,L-leucine was added to the formulation.

Cysteaminine bitartrate was allowed to warm to room temperature for 30minutes before opening. 100 mg Cysteamine Bitartrate powder, 50 mg ofL-Leucine and 850 mg of Trehalose were added to 10 ml deionised water,to give a total solids concentration of 10% w/v. This was stirred untilfully dissolved. Batches 052#140 and 052#155 were scaled to produce a 2g batch size.

The solution was spray dried using a Buchi B290 spray dryer, fitted witha high-efficiency cyclone and a Buchi two-fluid nozzle. Full spraydrying conditions are given in Table 5 below.

TABLE 5 Spray Drying Conditions Aspirator 100% Liquid Feed Rate 2ml/minute Atomisation Pressure 5.5 bar Inlet temperature 184° C. Outlettemperature 78° C.

Following spray drying, the moisture content of the product was reducedfurther by secondary vacuum drying, at ambient temperature, overnight.The final product was then stored in a sealed glass vial prior tocapsule filling. The solutions spray dried are summarised in Table 6below.

TABLE 6 Spray drying of Cysteamine Bitartrate formulations containingL-leucine Particle size analysis Solu- tion Weight of Weight WeightVolume of Spray dried Num- Cysteaminine of of L- deionised powder berBitartrate Trehalose Leucine water reference 1 100 mg 850 mg 50 mg 10 ml052#121 2 100 mg 900 mg 0 mg 10 ml 052#122 3 200 mg 1700 mg 100 mg 20 ml052#140 4 200 mg 1700 mg 100 mg 20 ml  052#155* *Collected as twobatches of approximately 1 g

Particle size analysis was performed using a SympaTec HELOS particlesize analyser with a RODOS disperser. Approximately 50 mg of formulationwas fed into the hopper. Dispersal was achieved using compressed air ata pressure of 2 bar. All instrument settings are detailed on theparticle size analysis reports in appendix 1 (data not shown).

Aerodynamic Particle Size Analysis by Andersen Cascade Impactor

The aerodynamic particle size of the spray dried powder was determinedusing a Copley Scientific 8 stage Andersen cascade impactor (ACI) fittedwith a 60 l/minute pre-separator and stages-1 to 6. The method was asdescribed in the US Pharmacopiea 29 general chapter <601>, and theEuropean Pharmacopeia 5.1. 2.9.18 (procedure for dry powder inhalers).

The following parameters were used:

Dose: 2× capsules

Capsules: Qualicaps HPMC standard size 3

Device: Plastiape, 3444, COQ, 23970000AA

Plate Coating: None

Airflow: Approximately 60 L/min (determined as a 4 KPa pressuredifferential across the device).

Actuation Time: Approximately 4 seconds (determined by airflow to equateto a volume of 4 litres)

Plate Washing: 0.1 M sodium phosphate buffer with EDTA, pH 8

Detection: UV at 412 nm using Ellmans reagent to provide a suitablechromophore

Cysteamine bitartrate concentration in the washings was measured at 412nm as described in section 3.6 below.

The mass of powder deposited at each stage was then calculated using theextinction coefficient determined in section 3.6. By analysing theamount of drug deposited on the various stages, it was then possible,using the dedicated Copley Scientific software, to calculate the FineParticle Dose (FPD), the Fine Particle Fraction (FPF), the Mass MedianAerodynamic Distribution (MMAD) and Geometric Standard Deviation (GSD)of the peptide particles collected.

The Fine Particle Dose (FPD) was defined as the quantity of drug in theprescribed dose of an inhaled product that is generally considered to beof a size capable of penetrating the lung during inhalation i.e.,respirable. This is usually considered to be about 5 microns or less.

The Fine Particle Fraction (FPF) was the FPD expressed as a percentageof the delivered dose.

Quantification of Cysteamine Bitartrate

The quantification of Cysteamine Bitartrate was conducted using aShimadzu UV-1650PC UV spectrometer. As Cysteamine Bitartrate has no UVchromophore Ellman's Reagent, 5,5-dithiobis(2-nitrobenzoic acid) wasused.

Preparation of Reagents

Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA.

Ellman's Reagent Solution: Dissolve 40 mg Ellman's Reagent in 10 mL

Reaction Buffer

Dissolve 34 mg of Cysteamine Bitartrate in 100 mL of Reaction Buffer toproduce a 1.5 mM solution.

Preparation of Standard Curve

Standards were prepared by dissolving Cysteamine Bitartrate in Reaction

Buffer at the following concentrations:

Volume of Amount of Reaction Cysteamine Final Standard Buffer mLBitartrate Concentration A 100 34 mg 1.5 mM B 5 25 mL of Standard A 1.25mM C 10 20 mL of Standard A 1.0 mM D 15 15 mL of Standard A 0.75 mM E 2010 mL of Standard A 0.5 mM F 25 5 mL of Standard A 0.25 mM G (Blank) 300 mL of Standard A 0.0 mMTable 7 Cysteamine Bitartrate Standards

A set of vials, each containing 50 μL of Ellman's Reagent Solution and2.5 mL of Reaction Buffer was prepared.

The assay solution or standard (250 μL) was added to the vials preparedin the previous step. The reagents were mixed and analysed on thespectrophotometer immediately. Absorbance was measured at 412 nm.

The values obtained from the standards were used to generate a standardcurve. The experimental sample concentration of Cysteamine Bitartrateare determined from this curve.

Results: Initial Studies on the Spray Drying Cysteamine Bitartrate withOleic Acid and Trehalose

Initial studies described in sections 3.1 confirmed that it was notpossible to produce a suitable dry powder by spray drying solutions ofcysteamine bitartrate containing oleic acid (with and withouttrehalose). Under all of the conditions used the resultant powderconsisted of a glassy, solid material that stuck to the walls of thecyclone and collection jar.

Improved results were obtained when oleic acid was removed from theformulation (see section 3.2). Removal of oleic acid resulted in theproduction of a fine, white powder (rather than a waxy solid). Howeverthe powder was still cohesive and had relatively poor flow properties.

Spray Drying of Cysteamine Bitartrate Formulations Containing Trehaloseand L-leucine

Powder properties improved when L-leucine was added to the feedsolution, resulting in fine white powders. Recoveries (yields) werehigh; in the range 50-83%. The spray dried powders had acceptablehandling properties, and could be easily recovered from the collectionvessel with minimal static charge. Formulations containing L-Leucine hada higher % yield and improved flow characteristics over those without.

Yields obtained from spray dried solutions containing L-leucine aresummarised in Table 8 below:

TABLE 8 Spray drying yields from formulations containing L-leucineWeight of powder Sample Reference recovered % Yield** 052#122  0.5 g 50052#121  0.7 g 70 052#140* 1.6 g 80 052#155* 1.7 g 83 **No residualmoisture accounted for *2 g batch sizeParticle Size Analysis of Spray Dried Cysteamine Bitartrate FormulationsContaining Trehalose and L-leucine

A summary of the particle size data for cysteamine bitartrateformulations containing trehalose and L-leucine are shown in Table 9.

TABLE 9 Particle size analysis (summary) X₁₀* X₅₀** X₉₀*** VMD****Sample (μm) (μm) (μm) (μm) 052#122 0.88 2.28 4.61 2.56 052#121 1.46 2.654.59 2.89 052#140 0.93 2.75 6.39 3.34  052#155A 0.74 1.92 4.30 2.28  052#155B 1.06 2.84 6.19 3.42 *10% of microparticles, by volume, belowthis figure **50% of microparticles, by volume, below this figure ***90%of microparticles, by volume, below this figure ****Volume mean diameterAerodynamic Particle Size Analysis by Anderson Cascade Impactor

A summary of the aerodynamic particle size data for spray dried batchesof cysteamine bitartrate, formulated with trehalose and L-leucine isshown in Table 10. Full particle size analysis reports are detailed inappendix 2 (data not shown).

TABLE 10 Aerodynamic Particle Size Gravimetric Gravimetric quantity ofquantity of Mass of formulation formulation API Capsule Capsule releasedreleased recovered A fill wt B fill wt from device, from device, fromthe FPD FPF Batch (mg) (mg) Capsule A Capsule B ACI (mg) (%) 052#121132.8 130.7 120.7 121.7 11.3 N/A* N/A* Run 1 052#121 114.8 119.6 105.0109.5 18.5 6.9 37.7 Run 2 052#122 84.0 75.2 54.8 54.9 11.2 3.0 27.0052#140 105.9 114.1 105.0 109.5 23.1 4.5 19.6 Run 1 052#140 96.9 100.990.2 93.4 25.9 5.6 21.5 Run 2 052#155 82.7 84.9 76.6 41.7 14.0 6.06 43.2Run 1 052#155 96.2 94.3 89.5 87.8 15.3 3.6 23.7 Run 2 *Not included dueto changes within the recovery process analytical method.

CONCLUSIONS

Cysteamine Bitartrate was successfully spray dried with trehalose andwith, or without L-leucine. In these studies, a formulation containingcysteamine bitartrate (10% w/w), trehalose (85% w/w) and L-Leucine(5%w/w) were superior in terms of powder recoveries, handling propertiesand drug loading into the capsules.

The improved powder handling characteristics of the cysteaminebitartrate/trehalose/leucine formulations were translated into anincrease in the Fine Particle Fraction (FPF), especially withformulations containing 5% Leucine.

Initial feasibility studies on DPI delivery confirm the spray driedpowders can be delivered using commercially available DPI's without alactose carrier. The initial feasibility studies used spray driedpowders provided with a FPF between 20% and 40% and a FPM between 3 and6.9 mg delivered from two capsules.

Example 2: Production of Spray Dried Cysteamine Bitartrate Formulationsfor In Vivo Testing Materials

Cysteamine bitartrate was supplied by NovaBiotics (Recordati 140514-1).All other reagents were analytical grade, supplied by Sigma.

Methods

Spray Drying of Cysteamine Bitartrate Formulations

Cysteamine bitartrate 5% (w/w), L-leucine 5% (w/w), mannitol 90% (w/w)(Batch 57#08a)

The cysteamine bitartrate powder was warmed to room temperature for 30minutes before opening. A solution containing 0.1 g cysteaminebitartrate powder, 0.1 g of L-Leucine and 1.8 g of mannitol was preparedin 20 ml deionised water, to give a total solids concentration of 10%w/v. This was stirred until fully dissolved.

The solution was spray dried using a Buchi B290 spray dryer, fitted witha high-efficiency cyclone and a Buchi two-fluid nozzle. Full spraydrying conditions are given in Table 11 below

TABLE 11 Spray drying conditions of Batch 57#08a Aspirator 100% LiquidFeed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature104° C. Outlet temperature 58° C.

Following spray drying, the powder was collected and stored in a glassvial using laboratory film and foil overwrapped within a protectiveenvironment with a % RH <10%

Cysteamine Bitartrate 10% (w/w), L-leucine 5% (w/w), mannitol 85% (w/w)(Batch 57#08b)

The cysteamine bitartrate powder was warmed to room temperature for 30minutes before opening. A solution containing 0.2 g cysteaminebitartrate powder, 0.1 g of L-Leucine and 1.7 g of mannitol was preparedin 20 ml deionised water, to give a total solids concentration of 10%w/v. This was stirred until fully dissolved.

The solution was spray dried using a Buchi B290 spray dryer, fitted witha high-efficiency cyclone and a Buchi two-fluid nozzle. Full spraydrying conditions are given in Table 12 below

TABLE 12 Spray drying conditions of Batch 57#08b Aspirator 100% LiquidFeed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature106° C. Outlet temperature 55° C.

Following spray drying, the powder was collected and stored in a glassvial using laboratory film and foil overwrapped within a protectiveenvironment with a % RH <10%

Placebo Batch Containing L-leucine 5% (w/w),) mannitol 95% (w/w) (Batch57#07)

A solution containing 0.1 g of L-Leucine and 1.9 g of mannitol wasprepared in 20 ml deionised water, to give a total solids concentrationof 10% w/v. This was stirred until fully dissolved.

The solution was spray dried using a Buchi B290 spray dryer, fitted witha high-efficiency cyclone and a Buchi two-fluid nozzle. Full spraydrying conditions are given in Table 13 below

TABLE 13 Spray drying conditions of Batch 57# 07 Aspirator 100% LiquidFeed Rate 2 ml/minute Atomisation Pressure 5.5 bar Inlet temperature100° C. Outlet temperature 64° C.

Following spray drying, the powder was collected and stored in a glassvial using laboratory film and foil overwrapped within a protectiveenvironment with a % RH <10%

Particle Size Analysis

Particle size analysis was performed using a SympaTec HELOS particlesize analyser with a RODOS disperser. Approximately 50 mg spray driedcysteamine bitartrate formulation was placed on the vibrating feeder andfed into the hopper. Dispersal was achieved using compressed air at apressure of 2 bar.

Analysis of Cysteamine Bitartrate Content in Spray Dried Powders

The quantification of Cysteamine Bitartrate was conducted using aShimadzu UV-1650PC UV spectrometer. As Cysteamine Bitartrate has no UVchromophore Ellman's Reagent, 5,5-dithiobis(2-nitrobenzoic acid) wasused to measure the sulphydryl group on the cysteamine.

Preparation of Reagents

Reaction Buffer: 0.1 M sodium phosphate, pH 8.0, containing 0.1 mM EDTA.

Ellman's Reagent Solution: Dissolve 40 mg Ellman's Reagent in 10 mLReaction Buffer

Dissolve 34 mg of Cysteamine Bitartrate in 100 mL of Reaction Buffer toproduce a 1.5 mM solution.

Preparation of Standard Curve

Standards were prepared by dissolving Cysteamine Bitartrate in Reaction

Buffer at the concentrations shown in Table 14:

TABLE 14 Cysteamine bitartrate standards Volume of Amount of ReactionCysteamine Final Standard Buffer mL Bitartrate Concentration A 100 34 mg1.5 mM B 5 25 mL of Standard A 1.25 mM C 10 20 mL of Standard A 1.0 mM D15 15 mL of Standard A 0.75 mM E 20 10 mL of Standard A 0.5 mM F 25 5 mLof Standard A 0.25 mM G (Blank) 30 0 mL of Standard A 0.0 mM

A set of vials, each containing 50 μL of Ellman's Reagent Solution and2.5 mL of Reaction Buffer was prepared.

The assay solution or standard (250 μL) was added to the vials preparedin the previous step. The reagents were mixed and analysed on thespectrophotometer immediately. Absorbance was measured at 412 nm.

The values obtained from the standards were used to generate a standardcurve. The experimental sample concentration of cysteamine bitartrateare determined from this curve.

Analysis of Cysteamine Content in Feed Solutions and Spray Dried Powders

The cysteamine bitartrate content was measured in each of the feedsolutions used to produce the two spray dried batches. A 100 μL aliquotof each solution was diluted into 10 ml of DI water to produce asolution that fell within the linear region of the standard curve. Thesamples were analysed as described in section 3.3.2 and cysteaminebitartrate concentration determined.

The cysteamine bitartrate content was measured in the two spray driedformulations. A 50 mg sample of each powder was diluted into 0.5 ml DIwater. A 100 μL aliquot was diluted into 10 ml of DI water to produce asolution that fell within the linear region of the standard curve. Thesamples were analysed as described in section 3.3.2 and cysteaminebitartrate concentration determined.

Results and Discussion

Spray Drying of Cysteamine Bitartrate Formulations

All feed solution was successfully spray dried, resulting in a finewhite powder. Recoveries are summarised in Table 15 below:

TABLE 15 Recovery of spray dried cysteamine formulations Amount spraydried Amount recovered Yield Batch No (g) (g) (%) 57#08a       2 g 1.050 57#08b      2 g 0.75 38 57#07 (placebo) 2 g 1.1 55

Recoveries for batches were lower than anticipated, however this islikely to be due to the small batch size (2 g). All powders had goodhandling properties, however it was noticed that the 10% cysteamineformulation was slightly more cohesive than the 5% formulation.

Particle Size Analysis

A summary of the particle size data for all time points is shown inTable 16 and representative particle size distributions are shown inFIGS. 1-3.

TABLE 16 Particle size analysis (summary) X₁₀* X₅₀** X₉₀*** VMD****Batch (μm) (μm) (μm) (μm) 57#08a      0.85 4.51 8.58 4.73 0.90 4.34 7.954.48 0.90 4.41 8.27 4.75 57#08b     1.62 6.61 12.69 7.16 1.83 6.89 13.287.49 1.91 6.99 13.21 7.52 57#07(placebo) 1.29 4.2 7.99 4.66 1.37 4.278.09 4.73 2.68 4.49 7.38 6.72 *10% of microparticles, by volume, belowthis figure **50% of microparticles, by volume, below this figure ***90%of microparticles, by volume, below this figure ****Volume mean diameter

Examples of the size distributions obtained for each batch are shown inFIGS. 1-3.

Determination of Cysteamine Content in Feed Solution and in Spray DriedPowders

Both the spray dryer feed solution and the spray dried powders producedwere analysed for cysteamine content. The results obtained are shown inTable 17 below

TABLE 17 Cysteamine content in feed solutions and spray dried powdersTarget Measured Sample concentration concentration Batch 57#08a (feedsolution)  5% (w/v)  5.9% (w/v) Batch 57#08a (spray dried   5% (w/w) 5.9% (w/w) powder) Batch 57#08b (feed solution) 10% (w/v)  11.5% (w/v) Batch 57#08b (spray dried  10% (w/w) 11.7% (w/w) powder)

In all samples the measured concentration was higher than the expectedconcentration based on the theoretical content.

Example 3: Assessment of Efficacy of Lynovex (Cysteamine) Prep in aMouse IN Neutropenic Model of Pseudomonas Aeruginosa ATCC 27853 (LungBurden Model)

Chemicals

Animals were immunosuppressed/pre-conditioned with either 200 mg/kg or150 mg/kg cyclophosphamide. Lynovex, chemical name cysteamine, andvehicle were either prepared either as Lynovex and lactose vehicle, orLynovex and mannitol-based vehicle (both provided by Upperton (Uppertonproduct)). These were prepared for treatment and vehicle-control alonerespectively, and in combination. Tobramycin was prepared as aninhalation formulation in lactose. All treatments were administeredusing a Penn Century device. Phosphate buffered saline (PBS) andPseudomonas selective agar were required for bacterial tissue burden.

Animals

Male CD1 mice (n =6 for treatment groups, plus five in pre-treatmentgroup, totalling 35 mice) were used in this study. On day −4, the micewere immunosuppressed/pre-conditioned with 200 mg/kg cyclophosphamideintraperitoneally; and with 150 mg/kg cyclophosphamide intraperitoneallyon day-1. An infection was established with P. aeruginosa ATCC27853,with an inoculum of 5×10⁶ cfu/ml, administered intranasally in a volumeof 40 μl following anaesthetisation with a ketamine/xylazine anaestheticcocktail for 15 minutes, for the Lynovex prepared in lactose study, andan inoculum of 4×10⁶ for the Upperton Lynovex product.

Treatment. All treatments were administered intratracheally using a PennCentury device.

Lynovex (cysteamine) was administered at 1.5 mg alone, and incombination with lactose at the following concentrations: Lynovex 0.75mg+2.25 mg lactose powder, Lynovex 1.5 mg+1.5 mg lactose powder, Lynovex2.25 mg+0.75 mg, along with a vehicle only control of 3 mg lactose. Inaddition, Tobramycin at 188 μg/dose was administered, as an inhaledformulation which was mixed with lactose to aid measurement. Thetreatments were administered approximately 5 minutes after infection.

In a different study, Lynovex was administered at the following doses: 3mg 5% Lynovex and 3 mg 10% Lynovex. Lynovex in combination withTobramycin as follows: 3 mg 5% Lynovex+Tobramycin 0.188 mg in 1.5 mgvehicle, 3 mg 10% Lynovex+Tobramycin 0.188 mg in 1.5 mg Vehicle(mannitol-based, provided by Upperton) and a Tobramycin only control(0.188 mg/dose in lactose vehicle). The treatments were administeredonce approximately 10 minutes after infection.

Bacterial Burden in Tissue

The lung tissue burden of each animal, at the clinical end point of 24 hpost-infection, was determined. The lungs were homogenised in 2 ml PBS,serially diluted in PBS and plated onto Pseudomonas selective agarbefore quantification after 24-48 h at 37° C.

With the Lynovex/Lactose study, a variable infection was achieved in thelungs of the mice infected with P. aeruginosa ATCC27853. Intratrachealdosing with 0.188 mg of the inhalation formulation of Tobramycinresulted in a statistically significant reduction in lung burden whencompared with vehicle-treated mice (P=0.0097 Kruskal Wallis test) and5/6 animals cleared the infection to below the limit of detection.Intratracheal administration of 1.5 mg and 2.25 mg Lynovex also reducedthe lung burden compared to vehicle (P=0.0072 and P=0.0349 respectively)with 5/6 and 4/6 mice respectively clearing the infection to below thedetection limit (FIG. 4).

In the Lynovex study with the Upperton product, a robust infection wasachieved in the lungs of the mice infected with P. aeruginosa ATCC27853.Intratracheal dosing with 0.188 mg of the inhalation formulation ofTobramycin resulted in highly variable burdens with an average 1.61 log10 cfu/g reduction in lung burden when compared with vehicle-treatedmice (Kruskal Wallis test). Intratracheal administration of 3 mg of 5%or 10% Lynovex as monotherapy did not reduce the lung burden compared tovehicle. However, combining 5% or 10% Lynovex with 0.188 mg Tobramycinresulted in a decrease in burden compared to vehicle mice (P<0.0001 andP<0.0001, respectively). This reduction was compared to treatment withTobramycin alone (P<0.0001 for 5% Lynovex+Tobramycin and P<0.0015 for10% Lynovex+Tobramycin, Kruskal-Wallis test) (FIG. 5).

Additionally, mouse weights were recorded before and after infection.Mice treated with vehicle, Lynovex monotherapy or Tobramycin monotherapylost weight following infection. In contrast mice treated with theLynovex+Tobramycin combinations maintained weight after infectionindicating they remained relatively healthy post infection (FIG. 6).

It should be noted that the Tobramycin for dry powder inhalation wassuspended in lactose rather than mannitol. Suspension in lactose led toclumping of the powder resulting in some difficulties in delivery asmany of the Penn Century devices blocked during dosing. The Lynovexsuspensions were much easier to administer and all were deliveredwithout issues due to the delivery device becoming blocked. Whilst thereductions in burden in the combination therapy arms are impressive andsignificantly superior to Tobramycin monotherapy, the data from somemice treated with Tobramycin monotherapy could be suspect due to thedifficulty in delivery of the DPI. Even when animals with uncertainTobramycin treatment are censored the greatly enhanced efficacy of thecombination arms still remains.

What is claimed is:
 1. A method of treating lung disease comprisingadministering to a subject suffering from lung disease a compositioncomprising microparticles wherein the microparticles comprise asulphur-containing compound and a stabilizing agent, wherein thesulphur-containing compound is selected from the group consisting ofcysteamine or cystamine; or a pharmaceutically acceptable salt, hydrateor ester thereof, and combinations thereof, wherein the stabilizingagent is selected from the group consisting of trehalose and sugaralcohols, and wherein the microparticles have a particle size of about 1micron to about 8 microns.
 2. The method of claim 1, wherein thecomposition further comprises at least one additional pharmaceuticalagent.
 3. The method of claim 2, wherein the at least one additionalpharmaceutical agent is an antibacterial agent.
 4. The method of claim2, wherein the at least one additional pharmaceutical agent is selectedfrom the group consisting of antibiotics, mucolytic agents,vasodilators, antihypertensive agents, cardiovascular drugs and calciumchannel blockers.
 5. The method of claim 1, wherein the lung disease isa respiratory disease.
 6. The method of claim 5, wherein the respiratorydisease is selected from cystic fibrosis, chronic obstructive pulmonarydisease, chronic bronchitis, bronchiectasis, emphysema, chronicobstructive airways disease, chronic cough, common cold, influenza,hantavirus, pneumonia, or pleurisy.
 7. The method of claim 1, whereinthe microparticles have a particle size of about 2 microns to about 8microns.
 8. The method of claim 1, wherein the composition isadministered by inhalation or intranasally.
 9. The method of claim 1,wherein the microparticles comprise up to 30% w/w of thesulphur-containing compound.
 10. The method of claim 1, wherein themicroparticles comprise up to 25% w/w of the sulphur-containingcompound.
 11. The method of claim 1, wherein the microparticles comprisebetween about 5% w/w and 10% w/w of the sulphur-containing compound. 12.The method of claim 1, wherein the microparticles comprise up to 85% w/wof the stabilizing agent.
 13. The method of claim 1, wherein thecomposition additionally comprises leucine.
 14. The method of claim 13,wherein the composition comprises between 1% w/w and 10% w/w of leucine.15. The method of claim 1, wherein the sugar alcohol is mannitol. 16.The method of claim 1, wherein the composition is in the form of a drypowder.